A protein extraction kit (KeyGEN, Nanjing, China) was used to lyse the cells. Protein concentrations were measured with a BCA kit (Beyotime, Shanghai, China). The proteins were separated by SDS–PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and was then incubated with antibodies against AMPK, p-AMPK (Thr172), mTOR, p-mTOR (Ser2448), NF-κBp65, phospho-NF-κBp65 (Ser536), p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), p70S6 kinase, phospho-p70 S6 kinase (Thr389), AKT, phospho-AKT (Ser473), Smad2, phospho-Smad2 (Ser465/467), β-actin, Beclin-1, LC3B, SQSTM1/p62 (Cell Signaling Technology, Beverly, MA, USA), fibronectin, α-smooth muscle actin (α-SMA), GAPDH, and human collagen (type I) (Thermo Fisher Scientific, Rockford, IL, USA) at 4 °C overnight. The membrane was then incubated with a goat anti-rabbit secondary antibody or a horse anti-mouse secondary antibody (Cell Signaling Technology, Boston, MA, USA) at room temperature for 1 h. Finally, the signals were examined using a chemiluminescence kit (Bio–Rad, Hercules, CA, USA).
α smooth muscle actin αsma
Manufactured by Thermo Fisher Scientific
Sourced in United States
α-smooth muscle actin (αSMA) is a structural protein found in the contractile apparatus of smooth muscle cells. It is a widely used marker for the identification and characterization of smooth muscle cells in various tissues and cell types.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Endomucin, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Jagged1, by R&D Systems (2 mentions)
Notch1, by R&D Systems (2 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Bca kit, by Beyotime (1 mentions)
Horse anti mouse secondary antibody, by Cell Signaling Technology (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence kit, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Hmb45, by Agilent Technologies (1 mentions)
Vimentin, by Agilent Technologies (1 mentions)
Synaptophysin, by Agilent Technologies (1 mentions)
Desmin, by Agilent Technologies (1 mentions)
7 protocols using α smooth muscle actin αsma
1
Protein Expression Analysis by Western Blot
2
Immunohistochemical Profiling of Tumor Samples
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Surgically resected tumors and PDX were formalin fixed and embedded in paraffin. H&E staining was used for assessment of pathology. For immunohistochemistry (IHC), 5‐μm thick sections were treated with primary antibodies against human PD‐L1 (22C3; Dako), human MHC class I (Hokudo), human CD8 (Dako), human CD31 (Leica), human CD68 (Dako), human myeloperoxidase, α‐smooth muscle actin (α‐SMA; Thermo Fisher Scientific), mouse CD31 (Abcam), and mouse F4 80 (Cedarlane). Next, they were incubated with secondary antibodies at room temperature and treated with Vectastain ABC Kit (Vector Laboratories). 3,3′‐Diaminobenzidine reaction was visualized by peroxidase activity.
3
Comprehensive Immunohistochemical Profiling
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HE staining: All specimens were fixed by 10% neutral formalin, regularly dehydrated, paraffin-embedded, sectioned into 3-μm sections, and processed for HE staining. IHC staining: IHC staining was performed using the EnVision two-step strategy. Primary antibody information: CD117, HMB45, Desmin, synaptophysin, chromograninA, S100 protein, wide-spectrum cytokeratin, Ki67, and vimentin (DAKO); Bcl2, P53, CD56, CD57 (LEICA); muscle specific actin (MSA), CD34, H-caldesmon (LongIsland); SOX10, INI1, DOG-1 (Gene Tech); somatostatin receptor 2 (SSTR2), somatostatin receptor 5 (SSTR5) (Abcam); α-smooth muscle actin (α-SMA) (Thermo); ATRX (Sigma); calponin, Collagen type IV (MXB Biotechnologies); BRAF V600E (ZSGB Biotechnology). The dilutions, clone and sources of these antibodies were listed in Table 1 .
4
Immunofluorescence Analysis of Tumor Markers
5
Establishment of Cisplatin-Resistant NSCLC Cell Lines
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The NSCLC cell lines, A549 and HCC827, were obtained from the Cell Bank of China Academy of Sciences (Shanghai, China). To establish the cisplatin-resistant cell line, A549-R, A549 cells were treated with a stepwise increasing concentration of cisplatin (Sigma-Aldrich, USA) as previously described.29 (link),30 (link) The CAFs and normal fibroblasts (NFs) were isolated from NSCLC tissues and adjacent normal tissues by primary culture, as previously described, respectively.31 (link),32 (link) Both third-generation purified CAFs and NFs were further identified by detecting CAFs-specific markers, including α-smooth muscle actin (α-SMA; Thermo, USA), fibroblast activation protein (FAP; Thermo, USA), and ferroptosis suppressor protein 1 (FSP1; Abcam, China). The A549 and HCC827 cells were cultured in RPMI 1640 medium (GIBCO-BRL, USA) supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in humidified air with 5% CO2 at 37 °C. The coculture system was created using transwell membranes with 0.4 μm pores (Corning, USA) in a 6-well plate. The CAFs (1 × 105) were treated with 20 μM GW4869 (Sigma-Aldrich, China) for 24 hours and placed above the membranes. The A549 and HCC827 cells were placed below the permeable membranes. The cells were placed in the coculture system for 72 hours in humidified air with 5% CO2 at 37 °C.
6
Corneal Neovascularization in Knockout Mice
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At day 7 post-cauterization, the mice were sacrificed and flat-mount corneal specimens were prepared (WT, n = 5; KO, n = 5). Uninjured WT (n = 5) corneas were also used. CD31- immunohistochmeistry was performed on each sample to evaluate limbal vessel inward extension. The length was measured of CD31-labeled endothelial cell protrusion from the limbus towards the center in cryosection samples. KO mice (n = 44) and WT mice (n = 40). Sixteen, 15, and 13 KO corneas and 16, 14, and 10 WT corneas were processed for cryosectioning on days 3, 7, 14, respectively. CD31 immunohistochemistry was performed on each sample and the distance was measured between the leading tip of CD31-labeled vessels in the stroma and the anterior chamber angle. Mean values of their length on both sides of the cornea were used to evaluate the extent of neovascularization. The Mann–Whitney U test was used for data analysis. Five, 5, 5, and 5 KO corneas and 5, 5 5, and 5 WT corneas were processed for 5 μm thick paraffin-sections on days 3, 7, and 14, of cauterized and uninjured tissue, respectively. Samples were processed for immunohistochemistry to evaluate the expression level of VEGF-A, (R&D systems, Minneapolis, MN, USA) and α- smooth muscle actin (αSMA) (Thermo Fisher Scientific, Waltham, MA, USA).
7
Immunofluorescence Analysis of Tumor Markers
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Paraffin tumor sections were subjected to double immunofluorescence staining by sequential method.14 (link) Primary antibodies used were endomucin (1:100; Santa Cruz Biotechnology), α-smooth muscle actin (αSMA; 1:100; Thermo Scientific), Notch1 (1:50; R&D Systems), Dll4 (1:50; R&D Systems), and Jagged1 (1:50; R&D Systems). AlexaFluor 488 and AlexaFluor 568 (Thermo Scientific) conjugates were used for fluorescence staining. For quantification, an average of 8 to 10 images, taken under 20x magnification, and 3 tumors per group were analyzed. Area (%) was determined using Fiji24 (link) using an arbitrary threshold applied to all images.
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